In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Evaluation of the antimutagenic and anticarcinogenic effects of inulin in vivo

The incidence of colorectal cancer is growing worldwide. The characterization of compounds present in the human diet that can prevent the occurrence of colorectal tumors is vital. The oligosaccharide inulin is such a compound. The aim of this study was to evaluate the antigenotoxic, antimutagenic and anticarcinogenic effects of inulin in vivo. Our study is based on 3 assays that are widely used to evaluate chemoprevention (comet assay, micronucleus assay, and aberrant crypt focus assay) and tests 4 protocols of treatment with inulin (pre-treatment, simultaneous, post-treatment, and pre + continuous). Experiments were carried out in Swiss male mice of reproductive age. In order to induce DNA damage, we used the pro-carcinogenic agent 1,2-dimethylhydrazine. Inulin was administered orally at a concentration of 50 mg/kg body weight following the protocols mentioned above. Inulin was not administered to the control groups. Our data from the micronucleus assay reveal antimutagenic effects of inulin in all protocols. The percentage of inulin-induced damage reduction ranged from 47.25 to 141.75% across protocols. These data suggest that inulin could act through desmutagenic and bio-antimutagenic mechanisms. The anticarcinogenic activity (aberrant crypt focus assay) of inulin was observed in all protocols and the percentages of damage reduction ranged from 55.78 to 87.56% across protocols. Further tests, including human trials, will be necessary before this functional food can be proven to be effective in the prevention and treatment of colon cancer. © FUNPEC-RP.

Glutathione and glutathione peroxidase expression in breast cancer: An immunohistochemical and molecular study

The use of prognostic markers for breast cancer allows therapeutic strategies to be defined more efficiently. The expression of glutathione (GSH) and glutathione peroxidase (GPX) in tumor cells has been evaluated as a predictor of prognosis and response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results showed that high GSH expression was associated with tumors negative for the estrogen receptor (ER) (P<0.05), and GPX expression was associated with tumors negative for the progesterone receptor (PR) and patient mortality. Focusing on the 37 patients who received adjuvant chemotherapy/radiotherapy (Group I), high expression of GPX was associated with a high rate of patient mortality (P<0.05). The 19 patients who received only adjuvant chemotherapy (Group II) showed high expression of GSH in relation to metastasis (P<0.05). In addition, high levels of GPX expression were significantly associated with a shorter overall survival (P<0.05). To confirm this, the expression of precursor genes of GSH [glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS)] and the GPX gene was analyzed using quantitative PCR in cultured neoplastic mammary cells treated with doxorubicin. Doxorubicin treatment was able to eliminate tumor cells without alterations in the gene expression of GSS, but led to underexpression of the GCLC and GPX genes. Our results suggest that high levels of GPX may be related to the development of resistance to chemotherapy in these tumors, response to treatment and the clinical course of the breast cancer patients.

Atividade citotóxica, pró-apoptótica, genotóxica e mutagênica de nitensidina B em células tumorais cervicais humanas

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Atividade antiproliferativa e antineoplásica de flavonóides da espécie Brosimum acutifolium em modelo de glioblastoma in vitro

Dentre os tumores que acometem o sistema nervoso, o glioblastoma multiforme (GBM), destaca-se por seu alto grau de agressividade e baixo prognóstico, apresentando em média uma sobrevida de 15 meses a partir do diagnóstico. O presente estudo objetivou investigar a atividade antiproliferativa e antineoplásica de quatro flavonoides isolados da espécie Brosimum acutifolium (Huber), duas flavanas: 4’-hidroxi-7,8-(2”,2”-dimetilpirano) flavana (BAS-1) e 7,4’-dihidroxi-8,(3,3-dimetilalil)-flavana, (BAS-4); e duas chalconas: 4,2’-dihidroxi-3’,4’-(2”,2”-dimetilpirano)-chalcona (BAS-6) e 4,2’,4’-trihidroxi-3’-(3,3-dimetilalil)-chalcona (BAS-7), em glioblastoma C6 de rato in vitro. Nossos resultados mostraram boa atividade citotóxica para as flavanas (BAS-1, -4) e para a chalcona BAS-7, com IC50 menor que 100 μM em teste de viabilidade pelo MTT, já a chalcona BAS-6, não demonstrou atividade citotóxica nas concentrações testadas. Estes flavonoides mostram ser menos citotóxico para célula não neoplásica (glia), com grau de segurança maior para a BAS-4 e BAS-7, uma vez que apresentaram menor efeito citotóxico à célula não neoplásica e menores índices hemolíticos. A análise de migração celular mostrou que o tratamento com BAS-1, BAS-4 e BAS-7 em baixas concentrações foi efetivo em promover inibição da migração celular. Estes três flavonoides também foram muito promissores em inibir a formação e o crescimento de colônia, além de promover parada no ciclo celular, com substancial aumento na população SubG0 para o tratamento com BAS-1 e BAS-4 com 100 μM. As flavanas BAS-1 e BAS-4 também mostraram maior capacidade de promover a perda na integridade do potencial de membrana mitocondrial (ΔΨm) e aumento para marcação com anexina V, indicativo de que estas drogas promovem morte por apoptose. No entanto a análise por microscopia eletrônica demonstrou marcantemente no tratamento com a BAS-4 a presença de vacúolos autofágicos, sugestivo que o processo de morte neste tratamento ocorre tanto por apoptose quanto autofagia. Com base nestes resultados pode-se concluir que dos flavonoides testados a BAS-1, BAS-4 e BAS-7 possuem potencial como agente antineoplásico na terapia do GBM, sendo a BAS-4 a mais promissora de todas.

Desenvolvimento e caracterização físico-química e biofarmacêutica de nano e microemulsões lipídicas de uso intravenoso contendo metildiidrojasmonato

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy

Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy. Cancer Res; 72(23); 6200-8. (C) 2012 AACR.

A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.

The bacterial colicin active against tumor cells in vitro and in vivo is verotoxin 1.

We have identified verotoxin 1 (VT1) as the active component within an antineoplastic bacteriocin preparation from Escherichia coli HSC10 studied over two decades. Recombinant VT1 can simulate the toxicity of anticancer proteins (ACP), and the antineoplastic activity of ACP (and VT1) was abrogated by treatment with anti-VT1 antibody. Similarly, VT1 mimics the protective effect of ACP in a murine metastatic fibrosarcoma model. Prior immunization with VT1 B subunit prevents the effect of VT1 or ACP in this model. The activity of ACP against a variety of human ovarian cell lines was mimicked by VT1, and multidrug-resistant variants were significantly hypersensitive. Primary ovarian tumors and metastases contain elevated levels of globotriaosylceramide compared with normal ovaries, and overlay of frozen tumor sections showed selective VT binding to tumor tissue and the lumen of invading blood vessels. Our contention that VT1 could provide an additional approach to the management of certain human neoplasms is discussed.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Aspetti meccanicistici della coordinazione di [(PPh3)3Ru(CO)(H2)] com Timina Acido Acetico

Ruthenium complexes have proved to exhibit antineoplastic activity related to the interaction of metal ion with DNA nucleobases. It is indeed of great interest to provide new insights on theses cutting-edge studies, such as the identification of distinct coordinative modes of DNA binding sites. During the investigation on the reaction between [(PPh3)3Ru(CO)(H)2], 1, and the Thymine Acetic Acid (THA) as model for nucleobases, we identified an unstable monohapto hydride acetate complex 2, which rapidly evolves into elusive intermediates whose nature was evidenced by NMR spectra and DFT calculations. We obtained crystals of [(PPh3)2Ru(CO)(k1-THA)(k2-THA)] 17, and [Ru(CO)(PPh3)2(k2-N,O)-[THA(A)];(k1-O)[THA(B)]2 18, phosphine ligands assuming cis conformation. The thesis deals on the analogue reactions of 1 with acetic acid by varying different parameters and operating conditions. The reaction yields to the hydride dihapto-acetate [(PPh3)2RuH(CO)(k2-Ac)] 8 through the related meridian monohapto, by releasing of phosphine ligand. However, the reaction yields a mixture of compounds, in which the dihapto hydride complex 8 is prevailing in any cases and does not provide any disclosure for the proposed mechanistic aspects. The reaction with two equivalents of acetic acid, affords the complex [(PPh3)2Ru(CO)(k1-Ac)(k2-Ac)] 11, exhibiting mutual trans:cis locations in 2:1 ratio for the phosphine. Such evidence agrees with the results obtained DFT calculations in vacuo, whereas it is in contrast with those obtained with the THA. Therefore we can inferred that the products obtained from the latter reaction is intermolecularly ruled by the hydrogen binding interactions between the functions [-NH•••(O)C-] in the two coordinated thymine ligands.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.